Small nuclear RNAs (snRNAs) interact with each other and premessenger RNA (pre-mRNA) to establish the structure of the spliceosomal. An exciting complexity in the analysis of splicing is emerging: some spliceosomal snRNAs appear to undergo secondary structure changes during splicing. Why and how are snRNA rearrangements important for snRNA function is splicing? How are these rearrangements achieved and regulated? What factors influence the initiation and control of these conformational changes? We will address these questions by identifying and characterizing the interaction of U2 snRNP with other components of the splicing apparatus. Our hypothesis is: factors that interact with U2 snRNA or mediated the RNA structural interconversion reactions within the spliceosome as it assembles and functions play an essential role in the regulation, progression and accuracy of splicing reactions. We will functions play an essential role in the regulation, progression and accuracy of splicing reactions. We will 1) characterize components that interact with U2, snRNA during spliceosome as it assembles and splicing, 2) probe changes in snRNA secondary structure or binding of factors to snRNAs during splicing, and 3)study of topology of U2, Ut, and the pre-mRNA during splicing. We have identified factors that interact genetically with U2 snRNA. Double mutant strains with heat sensitive prp 5, 9, 11 or 21 alleles and certain U2 alleles (some having no phenotype in a wild type background), die at temperatures normally permissive for growth or either single mutant. We have isolated new mutations that are suboptimal U3 negative (sun), have associated temperature sensitive splicing defects, and are not in any known prp gene. We have identified suppressor of a cold sensitive U2 mutation (CUS, cold sensitive U2 suppressor) that define new genes that modulate U2 function. We will characterize in detail the interaction of these gene products with U2 snRNA. Using chemical probes that modify RNA in a manner sensitive to subtitle changes in RNA structure and chemical environment, we will describe secondary structures adopted by different individual snRNAs as they proceed through the splicing pathway. This approach will provide direct evidence for such changes as well as relating these changes to the events in splicing. Finally, using a simple method we developed for producing large amounts of circular RNA, we will study the topological relationships between U2, U6, and pre-mRNA during the splicing reactions. U2 snRNP binding to pre-mRNA is a critical early step in the splicing pathway, one which appears to contribute to the regulation of alternative splicing. In addition, U2 snRNA is required function will provide a second catalytic steps of the splicing reaction. Knowledge about yeast U2 snRNA function will provide a sound conceptual basis for understanding the function and regulation of the splicing apparatus in mammals.